This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8). For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the protocol. Alternatively, use the MinElute Reaction Cleanup Kit.

Important points before starting

The yellow color of Buffer QG indicates a pH 7.5.

Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.

Procedure

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.

IMPORTANT: Solubilize agarose completely.

After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.